The bacterial cell envelope is a very complex structure that serves to protect the organism from its surrounding environment. In Gram-negative bacteria, the cell envelope is comprised of three distinct layers. The innermost layer is the inner membrane and the outermost layer is the outer membrane (OM). Between these membranes is a very crowded aqueous environment known as the periplasm. The OM is an asymmetrical lipid bilayer with glycerophospholipid (PL) localized on the inner leaflet facing the periplasm and lipopolysaccharide (LPS) localized on the outer leaflet facing the extracellular environment. This asymmetry promotes strong lateral interactions between LPS molecules in the presence of divalent cations, resulting in decreased OM permeability. During conditions of membrane stress (e.g. exposure to detergents), PLs from the inner leaflet will flip to the outer leaflet of the OM. If PLs cannot be removed from the outer leaflet, OM integrity is lost resulting in increased sensitivity to various antimicrobials. Several systems responsible for outer membrane maintenance have been described. The recently identified and highly conserved maintenance of OM lipid asymmetry (Mla) is one such system. Currently, there is no biochemical evidence supporting the proposed function of the Mla system. This represents a major gap in our knowledge of how bacteria maintain OM asymmetry. The overall objective of this application is to establish biochemical evidence to further our understanding of the Mla system mechanism of action. I propose two separate but complementary approaches to define the molecular mechanisms involved in the function of the Mla system. (1) I will investigate the direction of PL transport between the different PL-binding Mla proteins. I hypothesize, similar to the transport pathway for LPS and lipoproteins, Mla transport of PLs is unidirectional. (2) In a complementary approach, I will also investigate the protein-protein interactions of the different Mla proteins. This would be beneficial for the understanding of how the Mla system functions to remove mislocalized PLs from the OM. Regardless of the outcome of these studies, the information gathered during the course of completion of these Aims will greatly aid our knowledge on the essential mechanisms of OM asymmetry.